Antcin K, an Active Triterpenoid from the Fruiting Bodies of

4, Roosevelt Road, Taipei, Taiwan. Δ Department of Food Science, Rutgers University, New Brunswick, New Jersey, United States. § Department of Biolo...
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Antcin K, an Active Triterpenoid from the Fruiting Bodies of Basswood-Cultivated Antrodia cinnamomea, Inhibits Metastasis via Suppression of Integrin-Mediated Adhesion, Migration, and Invasion in Human Hepatoma Cells Ya-Ling Huang,† Yung-Lin Chu,†,Ω Chi-Tang Ho,†,Δ Jing-Gung Chung,§ Chiao-I Lai,† Yu-Cheng Su,† Yueh-Hsiung Kuo,*,Π and Lee-Yan Sheen*,†,⊥,# †

Institute of Food Science and Technology, #Center for Food and Biomolecules, and ⊥National Center for Food Safety Education and Research, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, Taiwan Δ Department of Food Science, Rutgers University, New Brunswick, New Jersey, United States § Department of Biological Science and Technology, China Medical University, Taichung, Taiwan Π Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resourceσ, China Medical University, Taichung, Taiwan S Supporting Information *

ABSTRACT: Previous research demonstrated that the ethyl acetate extract from Antrodia cinnamomea suppresses the invasive potential of human breast and hepatoma cells, but the effective compounds are not identified. The main bioactive compounds of A. cinnamomea are ergostane-type triterpenoids, and the content of antcin K is the highest. The objective of this study was to evaluate the antimetastatic activity and mechanisms of antcin K purified from the fruiting body of basswood-cultivated A. cinnamomea on human liver cancer Hep 3B cells. The results showed that adhesion, migration, and invasion of Hep 3B cells were effectively inhibited by antcin K within 24 h of treatment. Antcin K not only reduced the protein expression and activity of MMP2 and MMP-9 but also down-regulated vimentin and up-regulated E-cadherin in Hep 3B cells. In depth investigation for the molecular mechanism revealed that antcin K could reduce the protein expression of integrin β1, β3, α5, and αv and suppress phosphorylation of FAK, Src, PI3K, AKT, MEK, ERK, and JNK. These results suggested that antcin K was able to inhibit the metastasis of human hepatoma cells through suppression of integrin-mediated adhesion, migration, and invasion. Coupled with these findings, antcin K has a good potential to reduce the risk of liver cancer metastasis. KEYWORDS: liver cancer metastasis, Antrodia cinnamomea, antcin K, epithelial mesenchymal transition (EMT), matrix metalloproteinases (MMPs)

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INTRODUCTION According to cancer mortality statistics, liver cancer is ranked in the top two and top six for men and women worldwide, respectively.1 Most patients are diagnosed with the disease at the terminal stage as the symptoms for early-stage liver cancer are not obvious. However, >90% of cancer patients die from metastasis and not from original cancer. Thus, metastasis is the most difficult problem in cancer therapy.2 Today, we have chemical, radiation, and surgery treatments in liver cancer therapy, but they are accompanied by some side effects such as causing cancer cell metastasis. Therefore, it is a reasonable strategy to use natural active compounds in antimetastasis or complementary therapies. Antrodia cinnamomea is a treasured mushroom in Taiwan and has been reported to have effects in liver and neuron protection, anti-inflammation, anticancer, and blood pressure modulation, and immune system regulation.3,4 Antcin K is the most abundant ergostane triterpenoid of the fruiting bodies of basswood-cultivated A. cinnamomea. Although antcin K has been demonstrated to inhibit cancer cell proliferation, there is no report of its antimetastasis effect in liver cancer cells. This study investigated the detailed molecular mechanisms on © XXXX American Chemical Society

antimetastasis properties of antcin K in human liver cancer Hep 3B cells. Cancer metastasis is a complex process, including adhesion, migration, and invasion. In the process, some changes of genes and proteins in cells, such as epithelial mesenchymal transition (EMT), will occur.5 EMT is the process of invasion and migration of cells with epithelial characteristics transforming to cells of Leydig through gene and protein regulation. Normally, the process of EMT can be divided into three distinct stages. First, the down-regulation of protein expression of E-cadherin, claudins, and occludins will destroy the intracellular tight linkage; then, the cytoskeleton will be rearranged and the migration ability of cells will be increased; finally, cells within the degradation ability of extracellular matrix and basal membrane will invade other tissues and organs by increased expression of matrix metalloproteinases (MMPs). In addition, the secreted MMPs will destroy the structure of extracellular matrix and cause the migration of tumor cells to the Received: December 8, 2014 Revised: April 26, 2015 Accepted: April 26, 2015

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DOI: 10.1021/jf5059304 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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antibodies were obtained from Cell Signaling Technology, Inc. (St. Louis, MO, USA). Cell Culture. Hep 3B cells were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS, 1.5 g/L sodium bicarbonate, 2.38 g/L HEPES, 1% nonessential amino acids, and 1% antibiotic−antimycotic (100 units/mL penicillin, 0.1 μg/mL streptomycin, and 0.25 μg/mL amphotericin B) at 37 °C in a 5% CO2 incubator.10 Determination of Cell Viability MTT Assay. Liver cancer cells at a concentration of 5 × 103 cells/well were seeded in 96-well plates and incubated for 24 h, followed by treatment with 0 (0.02% DMSO), 5, 10, 15, 20, and 40 μM antcin K and incubated for a further 24 h. At the end of the stipulated period, 100 μL of MTT solution (0.5 mg/mL) was added and incubated at 37 °C for 4 h. The resulting MTT formazan was dissolved in 100 μL of DMSO and the absorbance recorded at 570 nm using a PowerWave HT Bio-Tek microplate spectrophotometer (Winooski, VT, USA).11 Detections of Cell−Matrigel Adhesion Assay. Hep 3B cells with 5 × 104 cells/mL were seeded into 6-well plates and incubated for 24 h, and the medium was then removed. After two washings, the medium was changed to 2 mL of 0, 5, 10, 15, and 20 μM antcin K with 2% FBS and incubated in 5% CO2 at 37 °C for 24 h. At the same time, matrigel (10 μg/30 μL) was coated into 96-well plates, 20 μL of BSA (2%) was added, and the mixture was incubated in 5% CO2 at 37 °C for 1 h. Next, Hep 3B cells with 5 × 104 cells/mL were put into 96well plates with 0, 5, 10, 15, and 20 μM antcin K in 2% FBS medium for 1 h. After removal of medium and two washings with PBS, 100 μL of 0.5 mg/mL MTT in 2% FBS medium was added. The following steps are the same as described in the cell viability MTT assay.12 Wound-Healing Assay. Putting ibidi culture-insert on the battle of 24-well plates, and Hep 3B cells with 3.5 × 104 cells/70 μL/well were seeded into other space and incubated in 37 °C at 5% CO2 for 24 h. The next step was taking of fthe culture insert and adding 500 μL/ well 0, 5, 10, 15, 20 μM antcin K in non-FBS medium incubation. Three pictures were taken by 100× microscopy at 0, 24, and 48 h randomly. Data were analyzed by ImageJ software to show the cell migration percentage compared with the control group.13 Cell Migration Assay. One transwell was set into 1 mL of PBS in 24-well plates and the unleakage transwell chosen after 30 min. To open the permeability of the transwell, 200 μL of PBS was added into the transwell and incubated for 18 h. PBS was removed next, and 1 mL of 10% FBS was added into the bottle of a 24-well plate seeded with Hep 3B cells (2.5 × 104 cells/100 μL/well, non-FBS medium) into the transwell. Then it was treated with 100 μL/well 0, 5, 10, 15, and 20 μM antcin K within non-FBS medium. After incubating in 37 °C at 5% CO2 for 24 h, the cells were fixed by methanol in 15 min, dyed for 45 min by 2% crystal violet, and washed out by PBS. Finally, five pictures were taken by microscopy (200×), and cell number was analyzed by ImageJ software to demonstrate the ability of cell migration (cell migration percent of control).14 Matrigel Invasion Assay. Most steps were similar to the cell migration assay; the only different step was we would coat 4 μg/μL matrigel into the transwell to avoid bubble formation.15 Gelatin Zymography. Hep 3B cells (5 × 104 cells/mL) were seeded into 24-well plates at 37 °C in 5% CO2 and incubated for 24 h, and the medium was removed and washed out with PBS twice. After treatment with various doses of antcin K, the upper medium was taken and mixed with 2× sample buffer (0.125 M Tris-HCl, 20% glycerol, 10% SDS, and 0.32% bromophenol blue). It was then loaded into 0.1% gelatin and 10% SDS-PAGE, run 100 V within 2 h, and 1× renaturing buffer (2.5% Triton X-100) was used to wash the gel twice; the gel was put in developing buffer (50 mM Tris-base, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij 35, pH 7.5) fpr incubation at 37 °C in 24 h. It was then dyed with Coomassie blue solution (2.5% Coomassie blue, 10% acetic acid, and 50% methanol) in 20 min. Finally, the destaining solution (10% acetic acid and 30% methanol) was used to remove color. After the gel had dried, it was analyzed by ImageJ.16 Western Blotting. For Western blotting, the cells (5 × 104 cells/ 10 mL/dish) were seeded in 10 cm2 dishes for 24 h. After 24 h of incubation, the cells were treated with 10 mL of medium containing 0, 5, 10, 15, and 20 μM antcin K for 24 h. After 24 h of treatment, total

extracellular matrix. Therefore, the MMPs play an important role in the invasion process.6 Integrins are receptors located between the cell surface and adhesion. They are not only involved in adhesion of cell and extracellular matrix but also active in the downstream cell signaling transduction pathways. They are heterodimers constituted of α- and β-subunits by noncovalent bonds; the α-subunit has 18 types, and the β-subunit has 8 types.7,8 In addition, integrin αvβ3 has been shown to be overexpressed in multiple cancer cell metastasis processes. Some studies demonstrate that integrin αvβ3 can bind with urokinase plasminogen activator (uPA) receptor and transfer plasminogen into plasmin that will then activate pro-MMPs and lead to the degradation of extracellular matrix by MMPs. Integrin αvβ3 can directly bind to MMP-2 as well and help the degradation of MMP-2.9 FAK is the main controller of integrin-mediated cancer cell signaling transduction pathway. When FAK and integrin mix together to form a focal adhesion complex, the whole structure will be changed. The steroid receptor coactivator (Src) will phosphorylate FAK; the activated p-FAK will in turn activate the signaling transduction pathway of PI3K/AKT, MEK/ERK, and JNK and regulates the adhesion, migration, and invasion of cancer cells. Therefore, the protein expression and phosphorylation of FAK are usually recognized as one of the biomarkers of cancer metastasis.9

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MATERIALS AND METHODS

Chemicals and Reagents. Basswood-cultivated A. cinnamomea (BCRC930103) mycelial powder was provided by PO-ZO Co., Ltd. (Taipei, Taiwan). Ethyl acetate (EA) was added to 500 g of A. cinnamomea dry powder to a total volume of 4 L and stirred for 3 days. The extract was then decanted and solvent removed by a rotary evaporator at 50 °C. The extraction was repeated. A silica gel column was used to fractionate the extracted sample and consecutively eluted with 10, 15, 20, 30, 50, 70, and 100% EA/hexane. The fraction with 100% EA/hexane contained the highest amount of antcin K. The purity of antcin K was >90% as determined by an HPLC analysis. The yield of antcin K from A. cinnamomea is about 0.5% in dry weight. Bromophenol blue and sodium dodecyl sulfate were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Antibiotic− antimycotic, Dulbecco’s modified Eagle’s medium, fetal bovine serum, and nonessential amino acids were purchased from Invitrogen (Carlsbad, CA, USA). Immobilon-P membrane (PVDF transfer membranes for Western blotting), Immobilon Western chemiluminescent HRP substrate (ECL), Millicell hanging cell culture insert (transwell), and dimethyl sulfoxide (DMSO) were obtained from Merck Millipore (Billerica, MA, USA). BD Matrigel Matrix High Concentration was purchased from BD Biosciences (San Jose, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Brij 35, gelatin, bovine serum albumin (BSA), HEPES sodium salt, TritonTM X-100, trypan blue, Tween 20, crystal violet, and other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Forty percent acrylamide/bis (29:1) solution, dithiothreitol (DTT), ammonium persulfate (APS), Coomassie brilliant blue R-250, glycerol, Tris base, and prestained protein ladder were purchased from Bioman (Taipei, Taiwan). Culture inserts (for wound-healing assay) were obtained from ibidi (Munchen, Germany). PRO-PREP protein extraction solution was purchased from Boca Scientific (Boca Raton, FL, USA). Trypsin−EDTA was obtained from Caisson Laboratories (North Logan, UT, USA). Acetic acid and methanol were purchased from Macron Chemicals (Center Valley, PA, USA). Phosphatase inhibitor cocktail tablet and protease inhibitor cocktail tablet were purchased from Roche (Basel, Switzerland). An SDS-PAGE kit was purchased from Bionovas Biotechnology (Toronto, ON, Canada). Anti-β-actin antibody and other secondary B

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Figure 1. Effects of antcin K on cell viability and proliferation in Hep 3B cells. (A) Effect of antcin K on the cell viability in Hep 3B cells was determined by MTT assay. Data are expressed as the relative percentage to control (0.02% DMSO) and mean ± SD from three independent experiments (n = 3). (B) Effect of antcin K on the expression of proliferating cell nuclear antigen in Hep 3B cells was assessed by Western blotting. Protein levels are expressed as multiple of control (0.02% DMSO) by β-actin-normalized densitometry. Columns represent the mean ± SD from three independent experiments (n = 3).

Figure 2. Changes by antcin K on the cell−matrix adhesion, migration, and invasion in Hep 3B cells. (A) Effect of antcin K on the cell−matrix adhesion in Hep 3B cells was determined by MTT assay. Data are expressed as the relative percentage to control (0.02% DMSO) and mean ± SD from three independent experiments (n = 3). (B) Effect of antcin K on the cell migration in Hep 3B cells was measured by Boyden chamber assay. The cells that migrated to the lower surface of the filter were stained with crystal violet and were photographed under a light microscope at 200×. Columns represent the mean ± SD from three independent experiments (n = 3). (C) Effect of antcin K on the cell invasion in Hep 3B cells was measured by Matrigel invasion assay. The cells that penetrated through the Matrigel to the lower surface of the filter were stained with crystal violet and were photographed under a light microscope at 200×. Columns represent the mean ± SD from three independent experiments (n = 3). polyvinylidene fluoride membranes, blotted with specific primary antibodies, and then labeled by horseradish peroxidase-conjugated secondary antibody according to the manufacturer’s instructions. The membranes were performed using the enhanced chemiluminescence and EC3300 Corning UVP biospectrum AC system (Corning, NY, USA), and the relative density of each band after normalization for βactin was analyzed using ImageJ 1.45 software.18

cell extracts were prepared in protein extraction solution, which contains 1 mM phenylmethanesulfonyl fluoride, 1 mM ethylenediaminetetraacetic acid, 1 μM pepstatin A, 1 μM leupeptin, and 0.1 μM aprotinin. The cell lysates were sonicated and cleared by centrifugation, and the protein concentration in the lysates was measured according to Lowry’s method.17 Fifty micrograms of proteins was loaded over 8% SDS-PAGE gels, transferred to C

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Figure 3. Effects of antcin K on the activity and protein expressions of MMP-2 and MMP-9 in Hep 3B cells. (A, B) Changes by antcin K on the activity of MMP-2 and MMP-9 in Hep 3B cells were assessed by gelatin zymography. Columns represent the mean ± SD from three independent experiments (n = 3). (C, D) Changes by antcin K on the expression of MMP-9 and MMP-2 in Hep 3B cells were assessed by Western blotting. Protein levels are expressed as multiple of control (0.02% DMSO). Columns represent the mean ± SD from three independent experiments (n = 3).

Figure 4. Effects of antcin K on the protein expression of integrin family and FAK-related protein in Hep 3B cells. (A) Effects of antcin K on the expression of integrin β1, integrin β3, integrin α5, and integrin αv in Hep 3B cells were assessed by Western blotting. The experiments were repeated at least three times with similar results (n = 3). Protein levels are expressed as multiple of control (0.02% DMSO) by GAPDH-normalized densitometry. (B) Effects of antcin K on the expression of FAK, p-FAK (Tyr397), p-FAK (Tyr576/577), and p-FAK (Tyr925) in Hep 3B cells were assessed by Western blotting. The experiments were repeated at least three times with similar results (n = 3). Protein levels are expressed as multiple of control (0.02% DMSO) by GAPDH-normalized densitometry. Statistical Analysis. All results are reported as the mean ± SD, and the differences between the antcin K-treated and control groups were analyzed by one-way analysis of variance ANOVA and Duncan’s multiple-comparison tests (SAS Institute Inc., Cary, NC, USA) to determine significant differences among treatments, p < 0.05.

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0, 5, 10, 15, 20, and 40 μM antcin K for 24 h in Hep 3B cells showed that, except for the 40 μM antcin K treatment, there was no significant difference of antcin K treatment in Hep 3B cells compared with the vehicle control group (0.02% DMSO, viable cells = 100%, p < 0.05) (Figure 1A). Protein Expression of PCNA on Antcin K in Hep 3B Cells. After treatment with 0, 5, 10, 15, 20, and 40 μM antcin K for 24 h in Hep 3B cells, the protein expression of proliferating cell nuclear antigen (PCNA) was not significantly different from the control group (p < 0.05). Therefore, 0−20 μM antcin

RESULTS AND DISCUSSION

Effects of Antcin K on Cell Viability in Hep 3B Cells Measured by MTT Assay. The MTT assay was used to assess the nontoxic dose of antcin K in Hep 3B cells. Treatment with D

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Figure 5. Total and phosphorylated protein expression of FAK’s three downstream signaling transduction pathways, PI3K/AKT, MEK/ERK, and JNK. After incubation of the cells with 0−20 μM antcin K for 24 h, the expressed proteins were assessed by Western blotting. The experiments were repeated at least three times with similar results (n = 3), and GAPDH was used as loading control. Protein levels are expressed as multiple of control (0.02% DMSO) by GAPDH-normalized densitometry and shown on the bottom of each band.

K would not affect the cell proliferation (Figure 1B) or the cell viability of Hep 3B cells (Figure 1A). Effects of Antcin K on Cell Adhesion in Hep 3B Cells. Hep 3B cells were pretreated with antcin K for 24 h and then seeded on 96-well microplates with matrigel. After 1 h, the medium was taken off, and the cells unattached on matrix were removed. The MTT assay was used to analyze the viable cells attached on matrigel to determine whether antcin K could inhibit the adhesion ability of Hep 3B cells. From Figure 2A, treatment of Hep 3B cells with 10 μM antcin K for 24 h could significantly inhibit Hep 3B cells’ attaching on matrigel compared with the control group (0.02% DMSO, adhesion rate = 100%). Furthermore, the inhibition rates of 10, 15, and 20 μM antcin K were 10.9 ± 2.9, 9.3 ± 3.0, and 15.0 ± 0.5%, respectively. Effects of Antcin K on Cell Migration in Hep 3B Cells. As shown in Figure 2B and Supplementary data 2, a Boyden chamber assay was used to analyze the migration effect of antcin K in Hep 3B cells. It showed that Hep 3B cells treated with 10, 15, and 20 μM antcin K for 24 h inhibited the migration of Hep 3B cells (p < 0.05), and the situation was similar to that in Supplementary data 1. It demonstrated that antcin K has significant migration inhibitory ability in human liver cancer cells. Changes by Antcin K on Cell Invasion in Hep 3B Cells. Invasion is an important step of metastasis processing. Matrigel invasion assay was used to assess the effect of antcin K on invasion of Hep 3B cells in 24 h (Figure 2C and Supplementary data 3). It was found that 10 μM antcin K treatment significantly decreased Hep 3B cells invasion compared with control group (0.02% DMSO, invasion rate = 100%, p < 0.05), and the inhibitory rates of 10, 15, and 20 μM antcin K in Hep 3B cells were 34.7 ± 10.7, 52.4 ± 6.4, and 63.5 ± 0.7%, respectively. From the data of cell adhesion, wound-healing assay, and migration and invasion assays, antcin K not only decreased the adhesion of extracellular matrix but also inhibited the migration and invasion of Hep 3B cells in 24 h (Figure 2). Therefore, antcin K could be considered to decrease the activity

of metastasis in human liver cancer cells. However, there are many reasons for metastasis, so additional experiments were designed to clarify the detailed mechanism of antcin K inhibiting Hep 3B cell metastasis. Effects of Antcin K on the Activity and Protein Expressions of MMP-2 and MMP-9 in Hep 3B Cells. MMPs belong to a large family of proteases, which are calciumdependent and zinc-containing endopeptidases. They are capable of degrading the composition of extracellular matrix and basal membrane such as collagens, laminin, fibronectin, and proteoglycans.19 There are at least 20 types of MMPs known. MMP-2 and MMP-9 are considered the most related to the invasion of cancer cells.9,10 The activities of MMP-2 and MMP-9 were analyzed by gelatin zymography as shown in Figure 3A,B.20 It was found that 10, 15, and 20 μM antcin K could inhibit the activity of Hep 3B cells (p < 0.05). The protein expressions of MMP-2 and MMP-9 had the same trends as activity (Figure 3C,D and Supplementary data 4). The data showed that antcin K could inhibit the protein hydrolysis of extracellular matrix and metastasis ability in Hep 3B cells. In addition, some researchers have shown that EMT-related proteins have a close relationship with MMP-2 and MMP-9.21,22 Therefore, we inferred that antcin K might inhibit the metastasis ability of Hep 3B cells via decreasing MMP-2 and MMP-9 protein expressions. Then the EMT-related proteins were also regulated by MMP-2 and MMP-9. EMT-Related Protein Expression of Antcin K in Hep 3B Cells. From Figure 6A, Western blotting was used to investigate the protein expression of E-cadherin and vimentin. Compared with the control group (0.02% DMSO), Hep 3B cells treated with 10−20 μM antcin K for 24 h, the protein expression of E-cadherin increased but the protein expression of vimentin decreased significantly (p < 0.05). These data supported our hypothesis about EMT-related proteins regulating metastasis ability after antcin K treatment in Hep 3B cells. E

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Figure 6. Effects of antcin K on the expression of E-cadherin, Src, and vimentin in Hep 3B cells were assessed by Western blotting: (A) effects of antcin K on the expression of E-cadherin and vimentin in Hep 3B cells; (B) effects of antcin K on the expression of Src and p-Src (Tyr416) in Hep 3B cells. The experiments were repeated at least three times with similar results (n = 3). Protein levels are expressed as multiple of control (0.02% DMSO) by GAPDH-normalized densitometry.

Figure 7. Potential mechanism of antimetastasis effect of antcin K in human liver cancer Hep 3B cells. F

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Journal of Agricultural and Food Chemistry Protein Expression of Integrin of Antcin K in Hep 3B Cells. Integrin is the receptor protein that mediates cell adhesion, involving the interaction of extracellular matrix and regular migration and invasion of cancer cells. Previous studies have shown that some integrin subunits such as β1, β3, α1, α2, α3, α4, α5, and αv have been detected in Hep 3B cells.23,24 However, various expressions of integrin are related to basal membrane, extracellular matrix-remodeling, adhesion, migration, invasion, and growth of cancer cells, and it is involved in the metastasis of multiple cancers.24 In 2002, integrins β1, β3, α5 and αv were found in different liver tumors from liver cancer patients. In 80 cases of liver cancer patients, 77 patients had overexpression of integrin β1, 44 patients had overexpression of integrin αv, 15 patients had β3 overexpression, and 70 patients had overexpression of integrin α5. These findings demonstrate that integrin β1, β3, α5, and αv are indeed involved in the development and metastasis of liver cancer.23 In addition, Ly et al. showed that the expression of integrin α5β1 could regulate integrin αvβ3-mediated adhesion or migration.25 For specific and functional property, integrin α5β1 only binds with fibronectin (Fn) of the extracellular matrix, which is the major component of the vascular wall. Fransvea et al. indicated that the expression of integrin α5β1 could regulate the migration of the vascular wall.26 To determine whether antcin K inhibiting metastasis was linked with the integrin family in Hep 3B cells, Western blotting was used to assess the integrin expression on antcin K treatment in Hep 3B cells. From the results, 10 μM antcin K could significantly decrease the protein expression of integrin β1, β3, α5, and αv in Hep 3B cells (Figure 4A). The data could explain that the antimetastasis ability of antcin K might be through the regulation of integrin receptor proteins in Hep 3B cells. Protein Expression of FAK and Src of Antcin K in Hep 3B Cells. FAK is one of the major controlling proteins of adhesion. Migration and invasion are partially mediated by integrin and FAK regulation. Compared with the control group, the total protein expression of FAK after treatment with antcin K did not change in Hep 3B cells (Figure 4B), but the protein expression and phosphorylation of FAK of 15 and 20 μM antcin K treatment decreased significantly within FAK (Tyr397), FAK (Tyr576/577), and FAK (Tyr925). In addition, the phosphorylated protein expression of Src decreased after antcin K treatment, but the total protein expression of Src did not change (Figure 6B). These data indicated that p-Src and p-FAK could mediate the antimetastasis mechanism of antcin K in Hep 3B cells. Protein Expression of PI3K/AKT, MEK/ERK, and JNK of Antcin K in Hep 3B Cells. To confirm whether the expression of FAK downstream was changed like FAK and Src, the signaling transduction proteins PI3K/AKT, MEK/ERK, and JNK were investigated. As shown in Figure 5, when Hep 3B was treated with various concentrations of antcin K for 24 h, the total protein expressions of PI3K, AKT, MEK, ERK, and JNK were not changed compared with the control group, but the phosphorylated protein expressions decreased in dosedependent manners. This demonstrated that antcin K treatment could decrease the phosphorylation of protein expression of FAK’s three downstream signaling transduction pathways such as PI3K/AKT, MEK/ERK, and JNK. Our data proved that antimetastasis of antcin K was truly via the activation of PI3K/ AKT, MEK/ERK, and JNK. Those results were agreeable to our data of viability and PCNA (Figure 1). For example, PI3K/ AKT also controls the survival pathway.27 Antcin K treatment

changed only the phosphorylation protein expression of PI3K/ AKT, which meant that 20 μM antcin K indeed inhibited the metastasis of Hep 3B cells and had no toxicity effect in Hep 3B cells. From our results, the integrin family could interact with cellular molecular, transfer the extracellular signaling into cytosol, and then affect the expression of nuclei DNA and cytoskeleton. Moreover, the receptor tyrosine kinases (RTKs), β1 integrin cytoplasmic domains, integrin-linked kinase (ILK), and other adaptor proteins such as focal adhesion kinase (FAK) of cell membrane might interact with integrin, forming a focal adhesion complex. In addition, the Ras/Raf/MEK/MAPK signaling pathway could be involved in multiple functions such as cell proliferation, invasion, angiogenesis, extracellular matrix remodeling, and anti-apoptosis, which was regulated by the integrin−actin−RTK network.28 Antcin K is the most abundant triterpenoid from the fruiting bodies of basswood-cultivated A. cinnamomea. Most studies use the ethanol extracts of A. cinnamomea as material,29−32 and previous study showed that the ethanol extracts of A. cinnamomea could inhibit the metastasis of human lung cancer CL1-0 cells through decreasing the phosphorylated protein expression of FAK, PI3K/AKT, JNK, and p38.33 Our data demonstrated that antcin K acted through a similar mechanism as ethanol extracts of A. cinnamomea in antimetastasis of cancer treatment; it is therefore reasonable to assume that antcin K plays a major role in ethanol extracts of A. cinnamomea. In conclusion, antcin K might inhibit the activation of PI3K/ AKT, MEK/ERK, and JNK by decreasing the protein expression of integrind β1, β3, α5, and αv and activating FAK. Then antcin K decreased protein expression and activation of MMP-2 and MMP-9 to affect EMT (Figure 7) and inhibited the metastasis in human liver cancer Hep 3B cells.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary data 1−4. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jf5059304

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AUTHOR INFORMATION

Corresponding Authors

*(Y.-H.K.) Mail: China Medical University, Hsueh-Shih Road, Taichung, 404, Taiwan, Republic of China. E-mail: kuoyh@ mail.cmu.edu.tw. *(L.-Y.S.) Mail: No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan. Phone: 886-2-33664129. Fax: 886-2-23620849. E-mail: [email protected]. Author Contributions Ω

(Y.-L.C.) International Master’s Degree Program in Food Science, International College, National Pingtung University of Science and Technology, 1 Shuefu Road, Neipu, Pingtung 91201, Taiwan. With equal contribution to first author. Funding

The technical work was supported by the Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University. In addition, the research work was partially funded by the National Science Council (NSC 100-2313-B-002-036) and CMU under the Aim for Top University Plan of the Ministry of Education, Taiwan, and Taiwan Ministry of Health and Welfare Clinical Trial and G

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Journal of Agricultural and Food Chemistry

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Research Center of Excellence (MOHW104-TDU-B-212113002). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EMT, epithelial mesenchymal transition; MMPs, matrix metalloproteinases; uPA, urokinase plasminogen activator; ECM, extracellular matrix; PI3K, phosphatidylinositol 3-kinase; Src, steroid receptor coactivator; FAK, focal adhesion kinase; JNKs, c-Jun N-terminal kinases; ERKs, extracellular signalregulated kinases; MEK, mitogen-activated protein kinase/ERK kinase; PKB, protein kinase B; PCNA, proliferating cell nuclear antigen

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DOI: 10.1021/jf5059304 J. Agric. Food Chem. XXXX, XXX, XXX−XXX